The objective of this project is to define the mechanisms of action of those antitumor agents recognized by the Division of Cancer Treatment as meriting such study, and to use the information obtained as a basis for defining improved ways to use the agents in experimental and clinical chemotherapy. Study of mechanisms of action of the agent include (a) investigation of the metabolism of the agent and the identification of active metabolites, if any, and of degradation products and; (b) definition of the biochemical sites of action of the agent and, where multiple sites are involved, determination of the primary site of action. The agents to be studied will be selected from among those under development by the DCT or those that may become of interest during the period of this grant. Detailed study will be limited to those agents whose sites of action appear to fall within the metabolic areas that encompass the particular expertise of the investigators, i.e., nucleotide and nucleic acid synthesis. It is anticipated that a new agent of unknown mechanism will be subjected to biochemical investigation to determine the broad metabolic area in which it acts, and that those that appear to act on synthesis of nucleotides or nucleic acids will be selected for study in depth under this grant. The area in which these agents act will be examined by studying their effects on macromolecular synthesis (RNA, DNA and protein synthesis). Metabolic studies with radioactive precursors in tumor cells in culture and in vivo will be used to examine and establish effects of inhibitors on nucleotide and nucleic acid biosynthesis. Specific effects of new agents on nucleotide pools and on specific enzymes of purine and pyrimidine nucleotide and nucleic acid biosynthesis will be explored. Tumor cell lines resistant to known anticancer agents provide a valuable means of relating new agents to existing clinically useful agents. We will make use of a broad spectrum of such resistant cell lines in the proposed work.